Imaging agents incorporating a targeting drug and visualizing moiety are indispensable in medical diagnostics and are invaluable aids in pharmacological drug development (9, 10, 23). Fluorophores absorbing in the visible region and emitting in the visible and near-infrared (IR) have found increasing application in this area owing to their scanning accessibility, convenience of use and sensitivity to detection (29). 5- or 6-Carboxyfluorescein (5-FAM, 6-FAM) and other fluorescent visible and near-IR labels are typically conjugated to a drug or protein by direct reaction of a primary amine of the drug or protein with a labels activated group (e.g. succinimidyl esters (SE), isothiocyanates (ITC), sulfosuccinimidyl esters (SSE), tetrafluorophenyl esters (TFP), sulfodichlorophenol esters (SC), etc.), thus forming an amide bond between the label and drug/protein (14). However, in cases when the parent drug structure lacks a primary amine group, often a linker (3) between the drug and label or structural modification to the drug (27) is necessary for labeling. In general, amido links are preferable to esters which may be labile to hydrolysis in vitro or in vivo.
Bone-targeting nitrogen-containing bisphosphonate drugs (N-BPs) such as (1-hydroxy-2-pyridin-3-ylethane-1,1-diyl)bis(phosphonic acid) 1, [hydroxy(1H-imidazol-1-yl)methylene]bis(phosphonic acid), {1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), (3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), and (4-amino-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) are extensively used in the clinic to treat osteoporosis and other disorders of bone metabolism (20, 21). Some bisphosphonate drugs have been shown to inhibit metastasis in bone cancer, and also to exhibit an anti-neoplastic effect on bone tumors (2, 7, 19). Alkylidenebisphosphonate drugs α-substituted with an aminoalkyl or N-containing heterocyclic group have been shown to inhibit specifically one or more enzymes of the mevalonic pathway; in at least some cases, the nitrogen atom is sufficiently basic to be protonated at physiological pH. X-ray crystallographic and modeling studies suggest that interaction of this nitrogen with target enzyme active site moieties contributes significantly to inhibitory potency and thus to the efficacy of this class of anti-osteoporotic drugs (5, 8, 11, 12, 15, 18). In contrast, the bone affinity is almost solely determined by the bisphosphonate moiety itself (18, 21, 28). Bisphosphonates have the general structure:

Fluorescently labeled bisphosphonate drugs can be useful in improving understanding of drug bone distribution, cellular distribution, and cell absorption selectivity. The clinically significant but thus far poorly understood anti-metastatic and anti-tumor cell effects of some bisphosphonates offers a further rationale for developing such imaging probes. Recent reports of a small number of previously unidentified osteonecrotic onsets that may be linked to prolonged therapy with at least one bisphosphonate also suggest an urgent requirement for improved understanding of bisphosphonate drug distribution in bone tissues (17, 22).
A conjugate of (4-amino-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) with a near-IR fluorophore (Alexa Fluor® 488, commercially available from Molecular Probes, Inc.), attached to the drug by formation of a carboxamide link with the drug's ε-amino group, was recently described drug (25). In this process, formation of the amide link greatly reduces the basicity of the N atom, abolishing its ability to acquire a positive charge by protonation. A comparable acylation approach for conjugating heterocyclic N-BPs such as 1 (via its pyridine nitrogen) is not facile, and no fluorescently N-labeled versions of such compounds have been available to date, to the inventors' knowledge. The structure of 1-hydroxy-2-pyridin-3-ylethane-1,1-diyl)bis(phosphonic acid 1 is:
